Leishmanias are sandfly-transmitted pathogenic protozoa affecting millions of people in tropical and subtropical regions. These parasites appear to depend on active mitochondrial functions including mRNA processing by RNA editing, throughout their life cycle, thus making the RNA editing machinery a promising drug target. Little is known, however, about mitochondrial functions including RNA editing during the mammalian stage (amastigotes). This gap in knowledge will be addressed by the proposed pilot studies which will utilize in vitro promastigote-amastigote differentiation systems of Leishmania donovani and Leishmania mexicana amazonensis. We will test the hypothesis that at least some of the kDNA-encoded transcripts are developmentally regulated at the level of RNA editing. This hypothesis is based on our early observation that RNA editing for some components of respiratory Complex I (NADH dehydrogenase) is dispensable in promastigotes of Leishmania tarentolae (equivalent to insect stage) suggesting that these components are required at a different stage of the life cycle. The specific goals are: (1) to determine the sequences of the conserved region of the maxicircles in the two pathogenic species; (2) to determine the sequences of edited mRNAs in promastigotes of these species; (3) to establish batch cultures of axenic amastigotes and perform a comparison with promastigotes in respect to unedited and edited mRNA ratios and patterns of editing; (4) to compare levels of the enzymatic and auxiliary protein components of the RNA editing machinery at both stages using available antibodies. These studies will shed light on the poorly known aspects of Leishmania cell biology with a potential of discovery of novel regulatory mechanisms and drug targets. [unreadable] [unreadable] [unreadable]